Methods and compositions for RNAi mediated inhibition of gene expression in mammals

ABSTRACT

Methods and compositions are provided for modulating, e.g., reducing, coding sequence expression in mammals. In the subject methods, an effective amount of an RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is administered to a non-embryonic mammal, e.g., via a hydrodynamic administration protocol. Also provided are RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including academic and therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. §119 (e), this application claims priorityto the filing date of the U.S. Provisional Patent Application Serial No.60/307,411 filed Jul. 23, 2001 and U.S. Provisional Patent ApplicationSerial No. 60/360,664 filed Feb. 27, 2002; the disclosures of which areherein incorporated by reference.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of this invention is RNAi.

[0004] 2. Background of the Invention

[0005] Double-stranded RNA induces potent and specific gene silencingthrough a process referred to as RNA interference (RNAi) orposttranscriptional gene silencing (PTGS). RNAi is mediated byRNA-induced silencing complex (RISC), a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger.

[0006] RNAi has become the method of choice for loss-of-functioninvestigations in numerous systems including, C. elegans, Drosophila,fungi, plants, and even mammalian cell lines. To specifically silence agene in most mammalian cell lines, small interfering RNAs (siRNA) areused because large dsRNAs (>30 bp) trigger the interferon response andcause nonspecific gene silencing.

[0007] To date, the Applicants are not aware of any report of successfulapplication of RNAi technology to non-embryonic mammalian organisms.Demonstration that RNAi works in non-embryonic mammalian organisms wouldprovide for a number of important additional applications for RNAitechnology, including both research and therapeutic applications, and istherefore of intense interest.

[0008] Relevant Literature

[0009] WO 01/68836. See also: Bernstein et al., RNA (2001) 7: 1509-1521;Bernstein et al., Nature (2001) 409:363-366; Billy et al., Proc. Nat'lAcad. Sci USA (2001) 98:14428-33; Caplan et al., Proc. Nat'l Acad. SciUSA (2001) 98:9742-7; Carthew et al., Curr. Opin. Cell Biol (2001) 13:244-8; Elbashir et al., Nature (2001) 411: 494-498; Hammond et al.,Science (2001) 293:1146-50; Hammond et al., Nat. Ref. Genet. (2001)2:110-119; Hammond et al., Nature (2000) 404:293-296; McCaffrrey et al.,Nature (2002): 418-38-39; and McCaffrey et al., Mol. Ther. (2002)5:676-684; Paddison et al., Genes Dev. (2002) 16:948-958; Paddison etal., Proc. Nat'l Acad. Sci USA (2002) 99:1443-48; Sui et al., Proc.Nat'l Acad. Sci USA (2002) 99:5515-20.

[0010] U.S. patents of interest include U.S. Pat. Nos. 5,985,847 and5,922,687. Also of interest is WO/11092. Additional references ofinterest include: Acsadi et al., New Biol. (January 1991) 3:71-81; Changet al., J. Virol. (2001) 75:3469-3473; Hickman et al., Hum. Gen. Ther.(1994) 5:1477-1483; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff etal., Science (1990) 247: 1465-1468; and Zhang et al., Hum. Gene Ther.(1999) 10: 1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349.

SUMMARY OF THE INVENTION

[0011] Methods and compositions are provided for modulating, e.g.,reducing, coding sequence expression in mammals. In the subject methods,an effective amount of an RNAi agent, e.g., an interfering ribonucleicacid (such as an siRNA or shRNA) or a transcription template thereof,e.g., a DNA encoding an shRNA, is administered to a non-embryonicmammal, e.g., via a hydrodynamic administration protocol. Also providedare RNAi agent pharmaceutical preparations for use in the subjectmethods. The subject methods and compositions find use in a variety ofdifferent applications, including academic and therapeutic applications.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 provides expression constructs employed in the RNAiexperiments described below.

[0013]FIGS. 2A to 2D: RNA interference in adult mice. FIG. 2A)Representative images of light emitted from mice co-transfected with theluciferase plasmid pGL3-Control and either no siRNA (left), luciferasesiRNA (middle) or unrelated siRNA (right). A pseudocolor imagerepresenting intensity of emitted light (red most and blue leastintense) superimposed on a grayscale reference image (for orientation)shows that RNAi functions in adult mammals. Forty μg of annealed 21-mersiRNAs (Dharmacon) were co-injected into the livers of mice with the 2μg of pGL3-Control DNA and 800 units of RNasin (Promega) in 1.8 ml ofPBS in 5-7 seconds. Seventy two hours after the original injection, micewere anesthetized and given 3 mg of luciferin intraperitoneally 15 minprior to imaging. FIG. 2B) Summary of siRNA data. Mice receivingluciferase siRNA emitted significantly less light than untreatedcontrols. A one-way ANOVA analysis with a post hoc Fisher's test wasconducted. The untreated and unrelated siRNA groups were statisticallysimilar. FIG. 2C) pShh1-Ff1 (center) but not pShh1-Ff1 rev (right)reduced luciferase expression in mice compared to the untreated control(left). 10 μg of pShh1-Ff1 or pShh1-rev were co-injected with 40 μg ofpLuc-NS5B in 1.8 ml of PBS. FIG. 2D) Quantitation of pShh1 data. Animalswere treated according to NIH Guidelines for Animal Care and theGuidelines of Stanford University.

[0014]FIG. 3 provides a schematic representation of the constructsemployed in the morpholino phosporamidate antisense HCV inhibition assayperformed in the Experimental Section, below.

[0015]FIG. 4 provides background information of the mechanism ofantisense inhibitors.

[0016]FIGS. 5A to 5F provide graphical results of a morpholinophosporamidate antisense HCV inhibition assay performed according to thesubject invention.

DEFINITIONS

[0017] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0018] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromsomal DNAof the host cell. Another type of vector is an episomal vector, i.e., anucleic acid capable of extra-chromosomal replication in an appropriatehost, e.g., a eukaryotic or prokaryotic host cell. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors”. In the presentspecification, “plasmid” and “vector” are used interchangeably unlessotherwise clear from the context.

[0019] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, as applicableto the embodiment being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

[0020] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide ofthe present invention, including both exon and (optionally) intronsequences. A “recombinant gene” refers to nucleic acid encoding suchregulatory polypeptides, that may optionally include intron sequencesthat are derived from chromosomal DNA. The term “intron” refers to a DNAsequence present in a given gene that is not translated into protein andis generally found between exons. As used herein, the term“transfection” means the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer.

[0021] A “protein coding sequence” or a sequence that “encodes” aparticular polypeptide or peptide, is a nucleic acid sequence that istranscribed (in the case of DNA) and is translated (in the case of mRNA)into a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

[0022] Likewise, “encodes”, unless evident from its context, will bemeant to include DNA sequences that encode a polypeptide, as the term istypically used, as well as DNA sequences that are transcribed intoinhibitory antisense molecules.

[0023] The term “loss-of-function”, as it refers to genes inhibited bythe subject RNAi method, refers a diminishment in the level ofexpression of a gene when compared to the level in the absence of theRNAi agent.

[0024] The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein coding sequence results fromtranscription and translation of the coding sequence.

[0025] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0026] By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

[0027] As used herein, the terms “transduction” and “transfection” areart recognized and mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses adsRNA construct.

[0028] “Transient transfection” refers to cases where exogenous DNA doesnot integrate into the genome of a transfected cell, e.g., whereepisomal DNA is transcribed into mRNA and translated into protein.

[0029] A cell has been “stably transfected” with a nucleic acidconstruct when the nucleic acid construct is capable of being inheritedby daughter cells.

[0030] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct that isheterologously expressed in a cell.

[0031] As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control. For purposes of this invention, the terms“transformed phenotype of malignant mammalian cells” and “transformedphenotype ” are intended to encompass, but not be limited to, any of thefollowing phenotypic traits associated with cellular transformation ofmammalian cells: immortalization, morphological or growthtransformation, and tumorigenicity, as detected by prolonged growth incell culture, growth in semi-solid media, or tumorigenic growth inimmuno-incompetent or syngeneic animals.

[0032] As used herein, “proliferating” and “proliferation” refer tocells undergoing mitosis.

[0033] As used herein, “immortalized cells” refers to cells that havebeen altered via chemical, genetic, and/or recombinant means such thatthe cells have the ability to grow through an indefinite number ofdivisions in culture.

[0034] The “growth state” of a cell refers to the rate of proliferationof the cell and the state of differentiation of the cell.

[0035] “Inhibition of gene expression” refers to the absence (orobservable decrease) in the level of protein and/or mRNA product from atarget gene. “Specificity” refers to the ability to inhibit the targetgene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). For RNA-mediated inhibition in a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereofmultiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

[0036] Depending on the assay, quantitation of the amount of geneexpression allows one to determine a degree of inhibition which isgreater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell nottreated according to the present invention. Lower doses of administeredactive agent and longer times after administration of active agent mayresult in inhibition in a smaller fraction of cells (e.g., at least 10%,20%, 50%, 75%, 90%, or 95% of targeted cells). Quantitation of geneexpression in a cell may show similar amounts of inhibition at the levelof accumulation of target mRNA or translation of target protein. As anexample, the efficiency of inhibition may be determined by assessing theamount of gene product in the cell: mRNA may be detected with ahybridization probe having a nucleotide sequence outside the region usedfor the inhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0037] Methods and compositions are provided for modulating, e.g.,reducing, coding sequence expression in mammals. In the subject methods,an effective amount of an RNAi agent, e.g., an interfering ribonucleicacid (such as an siRNA or shRNA) or a transcription template thereof,e.g., a DNA encoding an shRNA, is administered to a non-embryonicmammal, e.g., via a hydrodynamic administration protocol. Also providedare RNAi agent pharmaceutical preparations for use in the subjectmethods. The subject methods and compositions find use in a variety ofdifferent applications, including academic and therapeutic applications.

[0038] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0039] In this specification and the appended claims, the singular forms“a,” “an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0040] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0041] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, representativemethods, devices and materials are now described.

[0042] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the componentsthat are described in the publications which might be used in connectionwith the presently described invention.

[0043] RNAi in Non-Embryonic Mammals

[0044] As summarized above, the subject invention provides methods ofperforming RNAi in non-embryonic mammals. In further describing thisaspect of the subject invention, the subject methods of RNAi innon-embryonic mammals are described first in greater detail, followed bya review of various representative applications in which the subjectinvention finds use as well as kits that find use in practicing thesubject invention.

[0045] Methods

[0046] As indicated above, one aspect of the subject invention providesmethods of employing RNAi to modulate expression of a target gene orgenes in a non-embryonic mammalian host. In many embodiments, thesubject invention provides methods of reducing expression of one or moretarget genes in a non-embryonic mammalian host organism. By reducingexpression is meant that the level of expression of a target gene orcoding sequence is reduced or inhibited by at least about 2-fold,usually by at least about 5-fold, e.g., 10-fold, 15-fold, 20-fold,50-fold, 100-fold or more, as compared to a control. In certainembodiments, the expression of the target gene is reduced to such anextent that expression of the target gene/coding sequence is effectivelyinhibited. By modulating expression of a target gene is meant altering,e.g., reducing, transcription/translation of a coding sequence, e.g.,genomic DNA, mRNA etc., into a polypeptide, e.g., protein, product.

[0047] The subject invention provides methods of modulating expressionof a target gene in a non-embryonic mammalian organism. By non-embryonicmammalian organism is meant a mammalian organism or host that is not anembryo, i.e., is at a stage of development that is later in time thanthe embryonic stage of development. As such, the host organism may be afetus, but is generally a host organism in a post natal stage ofdevelopment, e.g., juvenile, adult, etc.

[0048] In practicing the subject methods, an effective amount of an RNAiagent is administered to the host organism to modulate expression of atarget gene in a desirable manner, e.g., to achieve the desiredreduction in target cell gene expression.

[0049] By RNAi agent is meant an agent that modulates expression of atarget gene by a RNA interference mechanism. The RNAi agents employed inone embodiment of the subject invention are small ribonucleic acidmolecules (also referred to herein as interfering ribonucleic acids),i.e., oligoribonucleotides, that are present in duplex structures, e.g.,two distinct oligoribonucleotides hybridized to each other or a singleribooligonucleotide that assumes a small hairpin formation to produce aduplex structure. By oligoribonucleotide is meant a ribonucleic acidthat does not exceed about 100 nt in length, and typically does notexceed about 75 nt length, where the length in certain embodiments isless than about 70 nt. Where the RNA agent is a duplex structure of twodistinct ribonucleic acids hybridized to each other, e.g., an siRNA(such as d-siRNA as described in copending application serial No.60/377,704; the disclosure of which is herein incorporated byreference), the length of the duplex structure typically ranges fromabout 15 to 30 bp, usually from about 15 to 29 bp, where lengths betweenabout 20 and 29 bps, e.g., 21 bp, 22 bp, are of particular interest incertain embodiments. Where the RNA agent is a duplex structure of asingle ribonucleic acid that is present in a hairpin formation, i.e., ashRNA, the length of the hybridized portion of the hairpin is typicallythe same as that provided above for the siRNA type of agent or longer by4-8 nucleotides. The weight of the RNAi agents of this embodimenttypically ranges from about 5,000 daltons to about 35,000 daltons, andin many embodiments is at least about 10,000 daltons and less than about27,500 daltons, often less than about 25,000 daltons.

[0050] In certain embodiments, instead of the RNAi agent being aninterfering ribonucleic acid, e.g., an siRNA or shRNA as describedabove, the RNAi agent may encode an interfering ribonucleic acid, e.g.,an shRNA, as described above. In other words, the RNAi agent may be atranscriptional template of the interfering ribonucleic acid. In theseembodiments, the transcriptional template is typically a DNA thatencodes the interfering ribonucleic acid. The DNA may be present in avector, where a variety of different vectors are known in the art, e.g.,a plasmid vector, a viral vector, etc.

[0051] The RNAi agent can be administered to the non-embryonic mammalianhost using any convenient protocol, where the protocol employed istypically a nucleic acid administration protocol, where a number ofdifferent such protocols are known in the art. The following discussionprovides a review of representative nucleic acid administrationprotocols that may be employed. The nucleic acids may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Jet injection may alsobe used for intra-muscular administration, as described by Furth et al.(1992), Anal Biochem 205:365-368. The nucleic acids may be coated ontogold microparticles, and delivered intradermally by a particlebombardment device, or “gene gun” as described in the literature (see,for example, Tang et al. (1992), Nature 356:152-154), where goldmicroprojectiles are coated with the DNA, then bombarded into skincells. Expression vectors may be used to introduce the nucleic acidsinto a cell. Such vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

[0052] For example, the RNAi agent can be fed directly to, injectedinto, the host organism containing the target gene. The agent may bedirectly introduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, etc. Methods for oral introductioninclude direct mixing of RNA with food of the organism. Physical methodsof introducing nucleic acids include injection directly into the cell orextracellular injection into the organism of an RNA solution. The agentmay be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of the agent may yield more effective inhibition; lowerdoses may also be useful for specific applications.

[0053] In certain embodiments, a hydrodynamic nucleic acidadministration protocol is employed. Where the agent is a ribonucleicacid, the hydrodynamic ribonucleic acid administration protocoldescribed in detail below is of particular interest. Where the agent isa deoxyribonucleic acid, the hydrodynamic deoxyribonucleic acidadministration protocols described in Chang et al., J. Virol. (2001)75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al.,Science (1990) 247: 1465-1468; Zhang et al., Hum. Gene Ther. (1999)10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are ofinterest.

[0054] Additional nucleic acid delivery protocols of interest include,but are not limited to: those described in U.S. patents of interestinclude U.S. Pat. Nos. 5,985,847 and 5,922,687 (the disclosures of whichare herein incorporated by reference); WO/11092;. Acsadi et al., NewBiol. (1991) 3:71-81; Hickman et al., Hum. Gen. Ther. (1994)5:1477-1483; and Wolff et al., Science (1990) 247: 1465-1468; etc.

[0055] Depending n the nature of the RNAi agent, the active agent(s) maybe administered to the host using any convenient means capable ofresulting in the desired modulation of target gene expression. Thus, theagent can be incorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc., administration.

[0056] In pharmaceutical dosage forms, the agents may be administeredalone or in appropriate association, as well as in combination, withother pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0057] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0058] The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

[0059] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0060] Furthermore, the agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

[0061] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0062] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0063] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0064] Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the nature ofthe delivery vehicle, and the like. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means.

[0065] Administration of an effective amount of an RNAi agent to anon-embryonic mammalian host according as described above results in amodulation of target gene(s) expression, e.g., a reduction of targetgene(s) expression, as described above.

[0066] The above described methods work in any mammal, whererepresentative mammals of interest include, but are not limited to:ungulates or hooved animals, e.g., cattle, goats, pigs, sheep, etc.;rodents, e.g., hamsters, mice, rats, etc.; lagomorphs, e.g., rabbits;primates, e.g., monkeys, baboons, humans, etc.; and the like.

[0067] The above described methods find use in a variety of differentapplications, representative types of which are now described in greaterdetail below.

[0068] Utility

[0069] The subject methods find use in a variety of differentapplications, where representative applications include bothacademic/research applications and therapeutic applications. Each ofthese types of representative applications is described more fullybelow.

[0070] Academic/Research Applications

[0071] The subject methods find use in a variety of different types ofacademic, research applications, in which one desires to modulateexpression of one or more target genes (coding sequences) in a mammalianhost, e.g., to determine the function of a target gene/coding sequencein a mammalian host. The subject methods find particular use in“loss-of-function” type assays, where one employs the subject methods toreduce or decrease or inhibit expression of one or more targetgenes/coding sequences in a mammalian host.

[0072] As such, one representative utility of the present invention isas a method of identifying gene function in a non-embryonic mammal,where an RNAi agent is administered to a mammal according to the presentinvention in order to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics using the subject methods determines the function ofuncharacterized genes by administering an RNAi agent to reduce theamount and/or alter the timing of target gene activity. Such methods canbe used in determining potential targets for pharmaceutics,understanding normal and pathological events associated withdevelopment, determining signaling pathways responsible for postnataldevelopment/aging, and the like. The increasing speed of acquiringnucleotide sequence information from genomic and expressed gene sources,including total sequences for mammalian genomes, can be coupled with useof the subject methods to determine gene function in a live mammalianorganism. The preference of different organisms to use particularcodons, searching sequence databases for related gene products,correlating the linkage map of genetic traits with the physical map fromwhich the nucleotide sequences are derived, and artificial intelligencemethods may be used to define putative open reading frames from thenucleotide sequences acquired in such sequencing projects.

[0073] A simple representative assay inhibits gene expression accordingto the partial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product. The function of the target genecan be assayed from the effects it has on the mammal when gene activityis inhibited.

[0074] If a characteristic of an organism is determined to begenetically linked to a polymorphism through RFLP or QTL analysis, thepresent invention can be used to gain insight regarding whether thatgenetic polymorphism might be directly responsible for thecharacteristic. For example, a fragment defining the geneticpolymorphism or sequences in the vicinity of such a genetic polymorphismcan be employed to produce an RNAi agent, which agent can then beadministered to the mammal, and whether an alteration in thecharacteristic is correlated with inhibition can be determined.

[0075] The present invention is useful in allowing the inhibition ofessential genes. Such genes may be required for organism viability atonly particular stages of development or cellular compartments. Thefunctional equivalent of conditional mutations may be produced byinhibiting activity of the target gene when or where it is not requiredfor viability. The invention allows addition of an RNAi agent atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

[0076] In situations where alternative splicing produces a family oftranscripts that are distinguished by usage of characteristic exons, thepresent invention can target inhibition through the appropriate exons tospecifically inhibit or to distinguish among the functions of familymembers. For example, a hormone that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone.

[0077] Therapeutic Applications

[0078] The subject methods also find use in a variety of therapeuticapplications in which it is desired to modulate, e.g., one or moretarget genes in a whole mammal or portion thereof, e.g., tissue, organ,etc. In such methods, an effective amount of an RNAi active agent isadministered to the host mammal. By effective amount is meant a dosagesufficient to modulate expression of the target gene(s), as desired. Asindicated above, in many embodiments of this type of application, thesubject methods are employed to reduce/inhibit expression of one or moretarget genes in the host in order to achieve a desired therapeuticoutcome.

[0079] Depending on the nature of the condition being treated, thetarget gene may be a gene derived from the cell, an endogenous gene, apathologically mutated gene, e.g. a cancer causing gene, a transgene, ora gene of a pathogen which is present in the cell after infectionthereof. Depending on the particular target gene and the dose of RNAiagent delivered, the procedure may provide partial or complete loss offunction for the target gene. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells.

[0080] The subject methods find use in the treatment of a variety ofdifferent conditions in which the modulation of target gene expressionin a mammalian host is desired. By treatment is meant that at least anamelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition.

[0081] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

[0082] The present invention is not limited to modulation of expressionof any specific type of target gene or nucleotide sequence.Representative classes of target genes of interest include but are notlimited to: developmental genes (e.g., adhesion molecules, cyclin kinaseinhibitors, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2,CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FOR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g.,APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); andenzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hemicellulases, integrases, inulinases, invertases, isomerases, kinases,lactases, Upases, lipoxygenases, lyso/ymes, nopaline synthases, octopinesynthases, pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases); chemokines (e.g. CXCR4, CCR5), the RNA component oftelomerase, vascular endothelial growth factor (VEGF), VEGF receptor,tumor necrosis factors nuclear factor kappa B, transcription factors,cell adhesion molecules, Insulin-like growth factor, transforming growthfactor beta family members, cell surface receptors, RNA binding proteins(e.g. small nucleolar RNAs, RNA transport factors), translation factors,telomerase reverse transcriptase); etc.

[0083] Kits

[0084] Also provided are reagents and kits thereof for practicing one ormore of the above-described methods. The subject reagents and kitsthereof may vary greatly. Typically, the kits at least include an RNAiagent as described above.

[0085] In addition to the above components, the subject kits willfurther include instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

[0086] Hydrodynamic Administration of Naked RNA

[0087] Also provided by the subject invention are methods andcompositions for the in vivo introduction of a naked nucleic acid, e.g.ribonucleic acid, deoxyribonucleic or chemically modified nucleic acids(including, but not limited to, morpholino, peptide nucleic acids,methylphosphonate, phosphorothioate or 2′-Omethyl oligonucleotides),into the target cell of a vascularized organism, e.g. a mammal. Thesemethods of the subject invention are conveniently referred to as“hydrodynamic” methods.

[0088] In one embodiment of the subject methods, an aqueous formulationof a naked nucleic acid and an RNase inhibitor is administered into thevascular system of the organism. In many embodiments, the aqueousformulation also includes a competitor ribonucleic acid, e.g. anon-capped non-polyadenylated ribonucleic acid. In yet otherembodiments, codelivery of DNA capable of being transcribed into the RNAmolecule with candidate modulatory agents is performed without an RNaseinhibitor or competitor ribonucleic acid, where the modulatory agent andthe DNA may or may not be delivered as a single composition. The subjectmethods find use in a variety of different applications, including bothresearch and therapeutic applications, and are particularly suited foruse in the in vivo delivery of a ribonucleic acid into a hepatic cell,e.g. for liver targeted in vivo delivery of nucleic acids.

[0089] In further describing this aspect of the subject invention, thesubject methods will be described first followed by a description ofrepresentative applications in which the subject methods find use andkits for use in practicing the subject methods.

[0090] Methods

[0091] As summarized above, the subject invention provides a method forthe in vivo introduction of a nucleic acid, e.g. a ribonucleic acid,into a target cell present in a vascularized multi-cellular organism. Byin vivo introduction is meant that, in the subject methods, the targetcell into which the nucleic acid is introduced is one that is present inthe multi-cellular organism, i.e., it is not a cell that is separatedfrom e.g. removed from, the multi-cellular organism. As such, thesubject methods are distinct from in vitro nucleic acid transferprotocols, in which a nucleic acid is introduced into a cell or cellsseparated from the multi-cellular organism from which they originated,e.g. are in culture. In other words, the subject methods are not methodsof in vitro nucleic acid transfer.

[0092] By introduction of the nucleic acid is meant that the nucleicacid, e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or anon-naturally occurring nucleic acid analog, is inserted into thecytoplasm of the target cell. In other words, the nucleic acid is movedfrom the outside of the target cell to the inside of the target cellacross the cell membrane.

[0093] By vascularized multi-cellular organism is meant a multi-cellularorganism that includes a vascular system. Multi-cellular organisms ofinterest include plants and animals, where animals are of particularinterest, particularly vertebrate animals that have a vascular systemmade up of a system of veins and arteries through which blood is flowed,e.g. in response to the beating of a heart. Animals of interest aremammals in many embodiments. Mammals of interest include; rodents, e.g.mice, rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs,cats; and primates, e.g. humans. In certain embodiments, themulti-cellular organism is a human. In other embodiments, themulti-cellular organism is a non-human mammal, e.g. a rodent, such as amouse, rat, etc.

[0094] As mentioned above, the subject methods are, in the broadestsense, suitable for introduction of nucleic acids into the target cellof a host. The term “nucleic acid” as used herein means a polymercomposed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides,or compounds produced synthetically (e.g. PNA as described in U.S. Pat.No. 5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids. The terms“ribonucleic acid” and “RNA” as used herein mean a polymer composed ofribonucleotides. The terms “deoxyribonucleic acid” and “DNA” as usedherein mean a polymer composed of deoxyribonucleotides.

[0095] The subject methods are particularly suited for use in thedelivery of a ribonucleic acid into a target cell of a multi-cellularorganism. As such, the methods will now be further described in terms ofthe delivery of ribonucleic acids. However, the following protocols arealso suitable for use in the delivery of other nucleic acids, e.g. DNAs(such as plasmid DNA), etc.

[0096] In practicing the subject methods, an aqueous composition of theribonucleic acid in which the ribonucleic acid is present as a nakedribonucleic acid is administered to the vascular system of themulti-cellular organism or host. In many embodiments, the naked RNAaqueous composition or formulation is administered to the vein of thehost, i.e. the naked RNA formulation is intravenously administered. Incertain embodiments, the naked RNA formulation is intravenouslyadministered to the host via high pressure injection. By high pressureinjection is meant that the aqueous formulation is intravenouslyintroduced at an elevated pressure, where the elevated pressure isgenerally at least about 20, usually at least about 30 mmHg. In manyembodiments, the elevated pressure ranges from about 10 to 50 mm Hg,where 40 to 50 mm Hg is often preferred. Methods of administeringaqueous formulations under high pressure, such as those described above,are described in the references listed in the relevant literaturesection, supra.

[0097] As mentioned above, the RNA or DNA that is to be introduced intothe target cell via the subject methods is present in the aqueousformulation as naked RNA. By “naked” is meant that the RNA is free fromany delivery vehicle that can act to facilitate entry into the targetcell. For example, the naked RNAs or DNAs delivered in the subjectmethods are free from any material that promotes transfection, such asliposomal formulations, charged lipids or precipitating agents, e.g.they are not complexed to colloidal materials (including liposomalpreparations). In addition, the naked RNAs of the subject invention arenot contained in a vector that would cause integration of the RNA intothe target cell genome, i.e. they are free of viral sequences orparticles that carry genetic information.

[0098] The naked RNAs that may be delivered via the subject inventionmay vary widely in length, depending on their intended purpose, e.g. theprotein they encode, etc. Generally, the naked RNAs will be at leastabout 10 nt long, usually at least about 30 nt long and more usually atleast about 35 nt long, where the naked RNAs may be as long as 20,000 ntor longer, but generally will not exceed about 10,000 nt long andusually will not exceed about 6,000 nt long. In certain embodimentswhere the naked RNA is an RNAi agent, as described above, the length ofthe RNA ranges from about 10 to 50 nt, often from about 10 to 40 nt, andmore often from about 15 to 30 nt, including 15 to 25 nt, such as 20 to25 nt, e.g., 21 or 22 nt.

[0099] The naked RNAs that may be introduced into a target cellaccording to the subject methods may or may not encode a protein, i.e.may or may not be capable of being translated into a protein uponintroduction into the target cell. In those embodiments where the nakedRNA is capable of being translated into a protein following introductioninto the target cell, the naked RNA may or may not be capped, it mayinclude an IRES domain, etc. However, in many particular protocols ofthis embodiment, the naked RNA is capped. Furthermore, the RNA in theseembodiments generally includes at least a polyadenylation signal, and inmany embodiments is polyadenylated, where the polyA tail, when present,generally ranges in length from about 10 to 300, usually from about 30to 50. Further description of the naked RNAs is provided infra.

[0100] As mentioned above, an aqueous formulation of the naked RNA isintravascularly, usually intravenously, administered to the host. In theaqueous formulations employed in the subject methods, an effectiveamount of the naked RNA is combined with an aqueous delivery vehicle. Byeffective amount is meant an amount that is sufficient to provide forthe desired amount of transfer into the target cell, e.g. to provide thedesired outcome, such as desired amount of protein expression. In manyembodiments, the amount of naked RNA present in the aqueous formulationis at least about 5 micrograms, usually at least about 10 micrograms andmore usually at least about 20 micrograms, where the amount may be asgreat as 10 milligrams or greater, but generally does not exceed about 1milligram and usually does not exceed about 200 micrograms.

[0101] Aqueous delivery vehicles of interest include: water, saline andbuffered media. Specific vehicles of interest include: sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, phosphate buffered saline, etc. The aqueous delivery vehiclesmay further include preservatives and other additives, e.g.antimicrobials, antioxidants, chelating agents, inert gases, nutrientreplenishers, electrolyte replenishers, divalent cations, such asmagnesium, calcium and manganese, etc. Of particular interest in manyembodiments is the use of buffered salt solutions arepseudophysiological.

[0102] A feature of certain embodiments of the subject methods is thatthe naked RNA is introduced into the vascular system of themulti-cellular organism in combination with an RNase inhibitor. By RNaseinhibitor is meant a compound or agent that at least reduces theactivity of, if not completely inactivates, an RNase activity in themulti-cellular organism. In many embodiments, the RNase inhibitor is aprotein inhibitor of RNase, where the human placental RNase inhibitor isof particular interest. The protein RNase inhibitor may be purified froma natural source or synthetically produced, e.g. via recombinanttechniques. Human placental RNase inhibitor may be obtained from avariety of different sources under a variety of different tradenames,where representative sources include: Promega, Inc., Strategene, Inc.,Fisher Scientific, Inc., and the like.

[0103] While the RNase inhibitor may, in certain embodiments, beadministered to the host in a composition separate from the aqueousnaked RNA composition, in many embodiments the RNase inhibitor ispresent in the aqueous naked RNA composition. The amount of RNaseinhibitor that is present in the aqueous composition is sufficient toprovide for the desired uptake of the naked RNA. Where the RNaseinhibitor is a protein inhibitor, the concentration of the inhibitor inthe aqueous composition that is introduced into the multi-cellularorganism during practice of the subject methods may range from about 4to 4,000 units, usually from about 400 to 4,000 units and more usuallyfrom about 400 to 1,500 units.

[0104] In certain embodiments, the naked RNA and RNase inhibitor areadministered in conjunction with a competitor RNA. By competitor RNA ismeant an RNA that is capable of serving as a competitive inhibitor ofRNase activity. In many embodiments, the competitor RNA is uncapped andnon-polyadenylated. By uncapped is meant that the competitor RNA lacksthe cap structure found at the 5′ end of eukaryotic messenger RNA, i.e.it lacks a 5′ 7 methyl G. By non-polyadenylated is meant that thecompetitor RNA lacks a polyA tail or domain of polyadenylation at its 3′end, as is found in eukaryotic messenger RNA. The length of thecompetitor RNA may vary, but is generally at least about 70 nt, usuallyat least about 200 nt and more usually at least about 1,500 nt, wherethe length may be as great as 10,000 nt or greater, but generally doesnot exceed about 3,500 nt and usually does not exceed about 1,500 nt.The concentration of competitor RNA in the aqueous composition issufficient to provide for the desired protection of the naked RNA (e.g.via competition for binding by RNase), and in many embodiments rangesfrom about 10 μg/ml to 10 mg/ml, usually from about 20 to 200 μg/ml andmore usually from about 40 to 150 μg/ml.

[0105] The subject methods result in highly efficient transfer of theadministered RNA into the cytoplasm of the target cell(s). The subjectmethods are particularly suited for transferring RNA into the cytoplasmof liver or hepatic cells and non-parenchymal cells in the liver. Assuch, in many embodiments the subject methods are in vivo methods ofachieving high level nucleic acid, e.g. RNA, transfer into hepatic cellsor liver tissue.

[0106] The nucleic acid that is introduced into the target cell via thesubject methods is short lived once inside the target cell. Depending onthe particular nature of the nucleic acid, the half life the nucleicacid following introduction via the subject methods generally rangesfrom about 30 sec to 10 days, usually from about 1 min to 24 hrs andmore usually from about 5 min to 10 hrs. As such, where the nucleic acidis an RNA encoding a protein of interest, protein expression followingintroduction via the subject method is transient, typically lasting fora period of time ranging from about 1 min to 3 days, usually from about5 min to 24 hrs. As such, in many embodiments of the subject methods,the subject methods are methods of providing for transient proteinexpression from a transgene, where protein expression is equal to RNAlifetime. Nonetheless, the protein expressed may have a longer lifetime,depending on the nature of the particular protein.

[0107] Utility

[0108] The subject methods find use in a variety of differentapplications in which the efficient in vivo transfer of a naked nucleicacid into a target cell is desired. Applications in which the subjectmethods find use include both therapeutic and research applications.Therapeutic applications of interest include gene therapy applications,vaccination applications, and the like. Research applications ofinterest include the production of animal models for particularconditions, e.g. RNA viral infections, the observation of geneexpression on phenotypes to elucidate gene function, etc. Otherapplications in which the subject invention finds use include thedevelopment of antisense, ribozyme and chimeraplasty (i.e. the repair ofgenes via RNA/DNA chimeras (see e.g. Yoon et al., Proc Natl Acad Sci USA(1996) 93(5):2071-6; Cole-Strauss et al., Science (1996)273(5280):1386-9; and Zhu et al., Proc Natl Acad Sci USA (1999)96(15):8768-73) therapeutics, as well as interfering RNA (RNA whosepresence in the cell prevents the translation of similar RNAs, (See e.g.Wianny et al., Nat Cell Biol (2000) 2(2):70-5; and SiQun et al., Nature(1998) 391: 806-811) therapeutics.

[0109] One type of application in which the subject methods find use isin the synthesis of polypeptides, e.g. proteins, of interest from atarget cell, particularly the transient expression of a polypeptide. Insuch applications, a nucleic acid that encodes the polypeptide ofinterest in combination with requisite and/or desired expressioncomponents, e.g. 5′ cap structures, IRES domains, polyA signals ortails, etc., is introduced into the target cell via in vivoadministration to the multi-cellular organism in which the target cellresides, where the target cell is to serve as an expression host forexpression of the polypeptide. For example, where the naked nucleic acidadministered by the subject methods is RNA, the RNA is an RNA that iscapable of being translated in the cytoplasm of the target cell into theprotein encoded by the sequence contained in the RNA. The RNA may becapped or uncapped, where when it is uncapped it generally includes anIRES sequence. The RNA also generally further includes a polyA tail,where the length of the polyA tail typically ranges from about 10 to300, usually from about 30 to 50 nt. Following in vivo administrationand subsequent introduction into the target cell, the multi-cellularorganism, and targeted host cell present therein, is then maintainedunder conditions sufficient for expression of the protein encoded by thetransferred RNA. The expressed protein is then harvested, and purifiedwhere desired, using any convenient protocol.

[0110] As such, the subject methods provide a means for at leastenhancing the amount of a protein of interest in a multi-cellularorganism. The term ‘at least enhance’ includes situations where themethods are employed to increase the amount of a protein in amulti-cellular organism where a certain initial amount of protein ispresent prior to practice of the subject methods. The term ‘at leastenhance’ also includes those situations in which the multi-cellularorganism includes substantially none of the protein prior to practice ofthe subject methods. As the subject methods find use in at leastenhancing the amount of a protein present in a multi-cellular organism,they find use in a variety of different applications, includingpharmaceutical preparation applications and therapeutic applications,where the latter is described in greater detail infra.

[0111] Therapeutic applications in which the subject methods find useinclude gene therapy applications in which the subject methods are usedto enhance the level of a therapeutic protein in the host organism andvaccination applications, in which the subject methods are used tovaccinate the host (or develop vaccines for delivery by other methods).As distinct from DNA based expression protocols, the subject RNA basedexpression protocols are uncomplicated by the need for promoter,enhancer, repressor and other regulatory elements commonly associatedwith eukaryotic genes. The subject methods may be used to deliver a widevariety of therapeutic nucleic acids which, upon entry into the targetcell, provide for the requisite enhanced protein level in the host.Therapeutic nucleic acids of interest include nucleic acids that replacedefective genes in the target host cell, such as those responsible forgenetic defect based diseased conditions, by encoding products that aresupposed to be provided to the host by these defective genes; nucleicacids which have therapeutic utility in the treatment of cancer; and thelike. Representative products involved in gene defect disease conditionswhose level may be enhanced by practicing the subject methods include,but are not limited to: factor VIII, factor IX, β-globin, low-densityprotein receptor, adenosine deaminase, purine nucleoside phosphorylase,sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembraneregulator, α-antitrypsin, CD-18, ornithine transcarbamylase,arginosuccinate synthetase, phenylalanine hydroxylase, branched-chainα-ketoacid dehydrogenase, fumarylacetoacetate hydrolase, glucose6-phosphatase, α-L-fucosidase, β-glucuronidase, α-L-iduronidase,galactose 1-phosphate uridyltransferase, and the like. Cancertherapeutic nucleic acids that may be delivered via the subject methodsinclude: nucleic acids that enhance the antitumor activity oflymphocytes by encoding appropriate factors, nucleic acids whoseexpression product enhances the immunogenicity of tumor cells, tumorsuppressor encoding nucleic acids, toxin encoding nucleic acids, suicidefactor encoding nucleic acids, multiple-drug resistance product encodingnucleic acids, ribozymes, DNA ribozymes, DNA/RNA chimeras, interferingRNA and antisense sequences, and the like.

[0112] An important feature of the subject methods, as described supra,is that the subject methods may be used for in vivo gene therapyapplications. By in vivo gene therapy applications is meant that thetarget cell or cells in which expression of the therapeutic gene isdesired are not removed from the host prior to practice of the subjectmethods. In contrast, the naked nucleic acid compositions areadministered directly to the multi-cellular organism and are taken up bythe target cells, following which expression of the encoded productoccurs.

[0113] As mentioned above, another therapeutic application in which thesubject methods find use is in vaccination of a host (as well asdevelopment of a vaccine to be delivered by other methods). In thesemethods, the naked nucleic acid, e.g. RNA, that is administered to thehost via the subject methods encodes a desired immunogen that, uponentry of the RNA into the target cell, is expressed and secreted toelicit the desired immune response. Vaccination methods in which nakednucleic acid are employed and in which the subject methods of nakednucleic acid delivery find use are further described in WO 90/11092, thedisclosure of which is herein incorporated by reference.

[0114] As mentioned above, the subject methods also find use in variousresearch applications. One research application in which the subjectinvention finds use is in the production of animal models of RNA virusinfection, where RNA viruses of interest include: HCV, HIV, influenza A,Hepatitis A, poliovirus, enteroviruses, rhinoviruses, aphthoviruses, andthe like. To produce such animal models, constructs are first providedthat include one or more regulatory elements from the RNA virus ofinterest operably linked to a reporter domain, e.g., a domain encoding adetectable product (such as luciferase, a fluorescent protein, etc.);etc. Alternatively, DNA constructs that can be transcribed in vivo intosuch RNA constructs may be employed. These constructs are thenadministered to a host, e.g., a mouse, according to the subject methodsto produce an animal model of an infection by the corresponding RNAvirus. As such, also provided are the animal models of RNA virusesproduced by the subject methods. A representative protocol for theproduction of an RNA virus animal model is provided in the experimentalsection infra.

[0115] The subject methods also find use in the delivery of RNAitherapeutic and/or research agents, including siRNA and shRNA, asdescribed more fully above and in the experimental section, below.

[0116] Also provided are methods of screening candidate modulatory,e.g., enhancing or inhibitory, agents using such animal models. Avariety of different types of candidate agents may be screened accordingto the subject methods.

[0117] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0118] Of particular interest in certain embodiments are antisensenucleic acids. The anti-sense reagent may be antisense oligonucleotides(ODN), particularly synthetic ODN having chemical modifications fromnative nucleic acids, or nucleic acid constructs that express suchanti-sense molecules as RNA. The antisense sequence is complementary tothe mRNA of the targeted gene, and inhibits expression of the targetedgene products. Antisense molecules inhibit gene expression throughvarious mechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

[0119] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about16 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

[0120] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0121] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993), supra, and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0122] Among useful changes in the backbone chemistry arephosphorodiamidate linkages, methylphosphonates phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. One example is the substitution of the ribose sugar with amorpholine. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

[0123] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0124] In such screening assays, the nucleic acid construct (e.g., theRNA or DNA construct described above) and the candidate agent areadministered to the host animal, the effect of the candidate agent onthe activity of the construct is observed, and the observed effect isrelated to the modulatory activity of the candidate compound. Thecandidate agent and nucleic acid construct may be administered to thehost according to the subject methods at the same or different times,where in certain preferred embodiments the two components areadministered to the host simultaneously, e.g., in the form of a singlefluid composition. Representative screening assays are provided in theexperimental section infra.

[0125] Another research application in which the subject methods finduse is the elucidation of gene function. In such methods, RNA having aparticular gene sequence is introduced via the subject methods and theeffect of the gene on the phenotype of the organism is observed.Benefits of using the subject methods for gene function researchapplications include the ability to express the genes without concernfor genetic regulatory elements. Other research applications in whichthe subject methods find use include, but are not limited to: the studyof ribozyme and antisense efficacy; the study of RNA metabolism, and thelike.

[0126] Kits

[0127] Also provided by the subject invention are kits for use inpracticing the subject methods of in vivo nucleic acid delivery to atarget cell, e.g. hepatic cells. The subject kits generally include anaked nucleic acid that is desired to be introduced into the target celland an RNase inhibitor. The subject kits may further include an aqueousdelivery vehicle, e.g. a buffered saline solution, etc. In addition, thekits may include a competitor RNA, as described supra. In the subjectkits, the above components may be combined into a single aqueouscomposition for delivery into the host or separate as different ordisparate compositions, e.g. in separate containers. Optionally, the kitmay further include a vascular delivery means for delivering the aqueouscomposition to the host, e.g. a syringe etc., where the delivery meansmay or may not be pre-loaded with the aqueous composition. In cases werethe reporter gene is transcribed in vivo from a DNA, RNase inhibitor andcompetitor RNA are not required.

[0128] In addition to the above components, the subject kits willfurther include instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g. a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium, e.g.diskette, CD, etc., on which the information has been recorded. Yetanother means that may be present is a website address which may be usedvia the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

[0129] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL

[0130] I. RNAi in Mammals

[0131] A. We co-delivered a 2 micrograms of plasmid that expresses aluciferase mRNA (pCMVGL3) mixed with 1.8 ml PBS, 1200 units of RNasinand 40 micrograms of competitor RNA along with the followingformulations:

[0132] 1) (Group 1 no RNA) 1.8 ml PBS as a untreated control;

[0133] 2) (Group 2 antisense RNA) 1.8 ml PBS mixed with 20 micrograms ofantisense orientation 21 mer RNA/DNA chimera with the sequence5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO:01) (deoxythymidilate residuesare indicated by dT, the remaining nucleotides are ribonucleotides); or

[0134] 3) (Group 3 RNAi) 1.8 ml PBS mixed with 20 micrograms ofantisense 21 mer described above annealed to 20 micrograms of its sensecomplement (with sequence 5′-CUUACGCUGAGUACUUCGAdTdT-3′)(SEQ ID. NO:02).

[0135] The oligonucleotides were kinased using adenosine triphosphateand T4 polynucleotide kinase. Each formulation (1-3) was tested by highpressure tail vein injection in 5 week old female Balb/c mice. At 5, 72and 96 hours post injection, light emitted as a result of luciferaseexpression was measured as described above. The results of thisexperiment are summarized in the table below. Numbers expressed asrelative light units. Group 1 Group 2 Group 3 Group 1 standard Group 2standard Group 3 standard no RNA error Antisense error RNAi error  3hours 1.11 × 10⁹ 2.05 × 10⁸ 1.29 × 10⁹ 7.90 × 10⁷ 7.90 × 10⁸ 3.54 × 10⁷72 hours 6.60 × 10⁶ 7.57 × 10⁵ 5.41 × 10⁶ 9.91 × 10⁵ 8.23 × 10⁵ 2.86 ×10⁵ 96 hours 3.41 × 10⁶ 4.50 × 10⁵ 2.72 × 10⁶ 5.25 × 10⁵ 4.61 × 10⁵ 6.77× 10⁴

[0136] The above results demonstrate that RNAi (group 3) caused thedestruction of luciferase RNA in the liver of an adult mammal. Thisdestruction resulted in a decrease in light emitted as a result ofluciferase activity when compared to animals that received no RNA orantisense oligonucleotide alone. To our knowledge, this is the firstdemonstration that RNAi is effective in an adult mammal. This methodprovides a model system to study the mechanism by which RNAi functionsin a mammal. It is also useful for the development and optimization ofRNAi based therapeutics. Furthermore, one need not codeliver theexpression plasmid with the modulating agent. One could also deliver amodulating agent targeting an endogenous gene.

[0137] B. Here, we test the ability of RNAi to suppress gene expressionin adult mammals. We find that synthetic small interfering RNAs (siRNAs)are potent inhibitors of gene expression in vivo. Furthermore,small-hairpin RNAs (shRNAs) are similarly effective. Notably, these RNAiagents can be delivered either as synthetic RNAs or transcribed in vivofrom DNA expression constructs. These studies indicate that RNAi can bedeveloped as a therapeutic tool and demonstrate that it can be employedwith conventional gene-therapy strategies.

[0138] 1. siRNAs

[0139] We modified existing hydrodynamic transfection methods J. Chang,L. J. Sigal, A. Lerro, J. Taylor, J Virol 75, 3469-73. (2001)) to permitefficient delivery of naked RNAs. Either an siRNA derived from fireflyluciferase or an unrelated siRNA were co-injected with a luciferaseexpression plasmid (construct description in FIG. 1). Luciferaseexpression was monitored in living animals using quantitative whole bodyimaging following injection of a luciferase substrate (4) and wasdependent on the amount of reporter plasmid injected and the time aftertransfection (data not shown). Representative animals are shown in FIG.2A. Quantification of these results is shown in FIG. 2B.

[0140] In each experiment, serum measurements of a co-injected plasmidencoding human α-1 antitrypsin (hAAT) (S. R. Yant, et al., Nat Genet 25,35-41. (2000)) served as an internal control to normalize transfectionefficiency and to monitor non-specific translational inhibition. Averageserum hAAT levels at 74 hours were similar in each group of animals.

[0141] Our results indicate specific siRNA-mediated inhibition ofluciferase expression in adult mice (p<0.0115); unrelated siRNAs werewithout effect (p<0.864). In 11 independent experiments, luciferasesiRNAs reduced luciferase expression (emitted light) by an average of81% (+/−2.2%).

[0142] 2. shRNA

[0143] Short hairpin RNAs (shRNAs) targeting firefly luciferase ofrenilla luciferase were synthesized by T7 polymerase in vitro runofftranscription. Co-transfection of these in vitro transcribed RNAs withpGL3-Control DNA resulted in reduced firefly luciferase expression inculture (Paddison et al, Genes Dev. 16(8):948-58 (2002)). In order totest whether these hairpin RNAs were functional in mice, wehydrodynamically transfected 40 μg of in vitro transcribed luciferaseshRNA (or as a control, renilla shRNA), 2 μg pGL3-Control DNA 2 μgpThAAT, 800 units of RNasin and 1.8 ml of PBS into mice. Light emittedfrom mice 72 hours after receiving firefly luciferase shRNAs was reducedby an average of 95% (+/−1.4%) compared to the untreated control. Lightemitted from mice receiving the renilla shRNA was reduced only slightly.Surprisingly, co-transfection of T7 transcription template DNA with aplasmid expressing the T7 polymerase protein did not lead to anyreduction in luciferase reporter activity in culture or in mice (datanot shown). Firefly Luciferase shRNA sequence (from 5′ to 3′)GGUCGAAGUACUCAGCGUAAGUGAUGUCCACUUAAGUGGGUGUUGUUUGUG (SEQ ID NO:11)UUGGGUGUUUUGGUU Renilla Luciferase shRNA sequence (from 5′ to 3′)GGGAUGGACGAUGGCCUUGAUCUUGUUUACCGUCACACCCACCACUGGGAG (SEQ ID NO:12)AUACAAGAUCAAGGCCAUCGUCUUCCU

[0144] The above results demonstrate that short in vitro transcribedhairpins also reduced luciferase expression in vivo.

3. Conclusion

[0145] The above data demonstrate that RNAi can downregulate geneexpression in adult mice.

[0146] C. Hepatitis C virus (HCV) is an RNA virus that infects 1 out of40 people worldwide and is the most common underlying cause for livertransplantation in the western world. To determine whether RNAi could bedirected against a human pathogen, several siRNAs were tested for theirability to target HCV RNAs in mouse liver. We used a reporter strategyin which HCV sequences were fused to luciferase RNA and RNAi wasmonitored by co-transfection in vivo. siRNAs targeting the HCV internalribosome entry site and core protein coding region failed to inhibitluciferase expression. In contrast, siRNAs targeting the NS5B region ofa chimeric HCV NS5B protein-luciferase fusion RNA reduced luciferaseexpression by 75% (+/−6.8%).

[0147] These results indicate the utility of using RNAi therapeuticallyto target important human pathogens.

[0148] D. From these data, it is clear that siRNAs are functional inmice. Functional shRNAs, which are equally effective in inducing genesuppression, can be expressed in vivo from DNA templates using RNApolymerase III promoters (Paddison et al., submitted). Expression of acognate shRNA (pShh1-Ff1) induced up to a 98% (+/−0.6%) suppression ofluciferase expression, with an average suppression of 92.8% (+/−3.39%)in three independent experiments (FIGS. 2C and 2D). An emptyshRNA-expression vector had no effect (data not shown). Furthermore,reversing the orientation of the shRNA (pShh1-Ff1 rev) insert abolishedsilencing, due to altered termination by RNA polymerase III andconsequent production of an improperly structured shRNA (Paddison etal., submitted). These data indicate that plasmid-encoded shRNAs caninduce a potent and specific RNAi response in adult mice. Furthermore,it demonstrates that this method of RNAi delivery can be tailored totake advantage of the significant progress that has been made in thedevelopment of gene-transfer vectors.

[0149] Existing gene therapy strategies depend largely upon the ectopicexpression of exogenous proteins to achieve a therapeutic result. Sinceits discovery, RNAi has held the promise of complementing thesegain-of-function approaches by providing a means for silencingdisease-related genes. Considered together, our results indicate thatRNAi can be induced in adult mammals using DNA constructs to direct theexpression of small hairpin RNAs. These studies demonstrate that thepresent invention provides viral and non-viral delivery systems forapplication, of therapeutic RNAi to a wide range of diseases.

[0150] II. Hydrodynamic Delivery of Naked RNA

[0151] A. Introduction

[0152] Unless otherwise noted, in all experiments RNAs and DNAs wereadded to the indicated amount of RNasin and brought to a final volume ofPBS equal to 1.4-1.8 milliliters. This solution was injected into thetail vain of the mice in 4-5 seconds. All RNAs used in these studieswere synthesized using an mMessage Machine kit and purified using anRNeasy kit (both from Qiagen Inc.). However, it should not be necessaryto purify the RNA and other purification methods exist that should alsowork. RNasin used in all the experiments listed here was native RNasinpurified from human placenta unless otherwise indicated (purchased fromPromega Inc.). For luciferase samples, at the indicated time, mice weregiven an intraperitoneal injection of luciferin (1.5 micrograms/grambody weight) and the light emitted from the mouse was measured.Background is ˜2×10² relative light units. Human factor IX samples wereanalyzed using an enzyme linked immunoassay.

[0153] B. Hydrodynamic Delivery of Naked RNA

[0154] RNAs coding for luciferase protein were injected into living micewith:

[0155] 1) no RNase inhibitor; or

[0156] 2) RNase inhibitor (called RNasin).

[0157] All RNA samples also contained an uncapped unpolyadenylated RNA(competitor RNA) that was included as a competitive inhibitor of RNaseactivity. Total RNA in each sample was adjusted to a total of 80micrograms with competitor RNA. As a negative control (described below)DNAs expressing the luciferase protein under the control of aprokaryotic promoter were also injected. At 3 and 6 hours mice weregiven an intraperitoneal injection of luciferin (the substrate for theluciferase enzyme) and the light emitted from the mouse was measured.

[0158] Results summarized in Table 1 TABLE 1 Number Nucleic of MiceRelative Light Units Acid Used (N) Formulation (RLU/ 5 min) Poly A RNA 1 4 units of RNasin  10 × 10⁶ Poly A RNA 1 400 units of RNasin 2.0 × 10⁷Poly A signal 1  4 units of RNasin 7.2 × 10⁴ RNA Template DNA 1 nonesignal at background

[0159] The above results show that:

[0160] Injected RNA is transfected into the liver of living mice.

[0161] Capped polyadenylated RNA with a poly A tail (Poly A RNA) istranslated in mouse livers because capped polyadenylated RNA gives astrong luciferase signal

[0162] Capped RNA with a poly A signal (Poly A signal RNA) is translatedin mouse livers but it gives a signal but it is about 100 fold lowerthan that seen with the RNA that has a poly A tail

[0163] The RNAs used in all the experiments described here weretranscribed from a bacterial promoter on a DNA plasmid. This promotershould not function efficiently in mammalian cells. The DNA template wasremoved after transcription using a DNase, however there is always theconcern that the signal seen could be the result of DNA contamination.To control for this, an amount of template DNA equivalent to that usedin the transcription was injected. If the signal is due to DNAcontamination then this sample should give a signal. However, no signalis seen from the DNA control.

[0164] It was also found that addition of an RNase inhibitor (calledRNasin) protects the RNA from degradation by serum nucleases, thusincreasing the observed signal, because addition of RNasin increased thesignal by 20 fold at the dose used.

[0165] From the above, the following conclusions are drawn. Hydrodynamicdelivery of naked RNA results in high level transfer of RNA into thelivers of living mice. Furthermore, capped and polyadenylated RNA worksbetter than RNA with a polyadenylation signal but no poly A tail,although both RNAs gave a signal. Addition of an RNase inhibitorprotected the RNA from degradation, resulting in a higher luciferasesignal. Finally, the signal seen with the injected RNA is not due to DNAcontamination.

[0166] C. Refinement of System

[0167] RNAs coding for luciferase protein were injected into living micewith 1) high or low doses of native or recombinant RNasin or 2) aftertreatment with RNase T1 which should destroy the RNA and abolish thesignal (negative control). All RNA samples also contained an uncappedunpolyadenylated competitor RNA such that the total amount of RNAinjected was 80 micrograms. Control DNAs expressing the luciferaseprotein under the control of a prokaryotic promoter were also injectedin indicated control reactions. At 3 and 6 hours mice were given anintraperitoneal injection of luciferin and the light emitted from themouse was measured. This experiment is largely to verify the results ofthe first experiment and to test which parameters are important. At thesix hour timepoint, one mouse that had been injected with RNA wassacrificed and its organs were removed to determine which organs expressluciferase.

[0168] The results are summarized in Table 2 TABLE 2 micro- RelativeLight Relative Light Relative Light grams Number Units (RLU/5 Units(RLU/5 Units (RLU/5 Nucleic Acid of RNA of Mice min) min) min) Used orDNA (N) Formulation 3 hours 6 hours 24 hours Poly A RNA 35 1  240 units1.8 × 10⁵ 1.1 × 10⁶ Background RNasin Native Poly A RNA 50 1  240 units1.6 × 10⁵ 5.4 × 10⁵ Background RNasin Native Poly A RNA 50 1  44 units5.5 × 10⁴ 1.9 × 10⁵ RNasin Native Poly A RNA 10 1  240 units 7.7 × 10⁴1.8 × 10⁵ RNasin Recombinant Poly A RNA 50 2 3000 units BackgroundBackground RNase T1 Template 2 1 none Background Background DNA

[0169] The above results demonstrate that:

[0170] The dose of RNasin alters the level of expression seen becauseincreasing doses of RNasin lead to increased levels of luciferaseactivity.

[0171] Both native and recombinant RNasin both protect the RNA.

[0172] When the RNA is destroyed with RNase, the signal is abolished,demonstrating that the RNA is responsible for the signal (negativecontrol).

[0173] When an amount of template DNA equivalent to that used in thetranscription is injected without DNase treatment, no signal is seen,demonstrating that the signal is not due to DNA contamination.

[0174] Liver is the only site of luciferase expression.

[0175] From the above, the following conclusions are drawn. RNasin doseeffects the level of expression. Both recombinant and native RNasinprotect the injected RNA. No signal was seen when template DNA wasinjected or when RNA was destroyed with RNase, demonstrating that signalis not the result of DNA contamination. Finally, liver is the only siteof luciferase expression.

[0176] D. Competitor RNA Enhances the Activity.

[0177] Luciferase activity from 20 micrograms of capped andpolyadenylated luciferase RNA was measured. Four conditions were testedin experiments similar to those described in experiments 1 and 2:

[0178] 1) 400 units of RNasin+competitor RNA;

[0179] 2) 40 units of RNasin with no competitor RNA;

[0180] 3) 800 units of RNasin with no competitor RNA;

[0181] 4) 1200 units of RNasin with no competitor RNA.

[0182] At 3, 6 and 9 hours mice were given an intraperitoneal injectionof luciferin and the light emitted from the mouse was measured.

[0183] The results are summarized in Table 3. TABLE 3 Micro-grams NumberAverage Average Average Competitor Units of of Mice (RLU/2 min) (RLU/2min) (RLU/2 min) RNA RNasin (N) 3 hours 6 hours 9 hours RLU 60 400 3 7.6× 10⁴ 1.7 × 10⁴ 3.5 × 10³ standard 3.5 × 10⁴ 4.2 × 10³ 9.6 × 10² errorRLU None 400 3 6.5 × 10³ 4.2 × 10³ 2.6 × 10³ standard 1.4 × 10³ 2.8 ×10³ 1.7 × 10³ error RLU None 800 3 6.2 × 10³ 8.7 × 10³ 2.0 × 10³standard 3.1 × 10⁴ 2.5 × 10³ 3.7 × 10² error RLU None 1200 3 7.6 × 10⁴2.2 × 10⁴ 7.4 × 10³ standard 5.4 × 10⁴ 1.6 × 10⁴ 4.5 × 10³ error

[0184] The above results demonstrate that:

[0185] RNasin dose alters the luciferase activity because increasingdoses of RNasin lead to increasing luciferase activity. The highest dose(1200 units of RNasin) gave the highest activity at all times tested.

[0186] The addition of competitor RNA enhanced the measured luciferaseactivity, because presence of the competitor RNA enhanced the luciferaseactivity. This effect was synergistic with the protective effect of theRNasin.

[0187] From the above results, the following conclusions are drawn.Addition of competitor RNA increases luciferase signal. Furthermore,increasing doses of RNasin lead to increasing levels of luciferaseactivity

[0188] E. Cap Independent Translation of Luciferase Using an InternalRibosome Entry Site.

[0189] In Eukaryotes, translation of RNAs into protein occurs by twodifferent mechanisms called cap dependent and cap independenttranslation. Cap independent translation requires a 5′ nontranslatedregion called an internal ribosome entry site (IRES). Several RNAviruses, such as hepatitis C virus (HCV), polio virus and hepatitis Autilize IRES sequences to carry out cap independent translation. Weoriginally developed the RNA transfection method described here with theidea that it could be used to make a small animal model system forstudying anti-HCV therapeutics. Transfection with IRES RNAs could alsobe used for mutagenesis studies designed to investigate sequenceelements necessary for efficient IRES function.

[0190] 1. Description of Experiment and Results:

[0191] The RNA HCVluc has the HCV IRES at the 5′ end and the luciferasegene followed by a poly A tail. 40 micrograms of HCVluc+40 micrograms ofcompetitor RNA+20 microliters of RNasin were injected into the tail vainof the mice. At 3 and 6 hours mice were given an intraperitonealinjection of luciferin and the light emitted from the mouse wasmeasured. Result: The HCV IRES was able to drive translation of theinjected HCV luciferase RNA fusion. Quantitation of the results issummarized in Table 4. TABLE 4 3 hours post injection 6 hours postinjection Average Relative Light 1.7 × 10⁵ 4.6 × 10⁴ Units StandardError 7.4 × 10⁴ 1.6 × 10⁴

[0192] F. Measurable serum concentrations of human factor IX (hFIX)protein can be produced and secreted upon injection of hFIX RNA.

[0193] Human factor IX protein is a blood clotting protein that is notproduced by some patients with hemophilia. The levels of this protein inserum can be easily measured using an enzyme linked immunoassay (ELISA).We chose to express this protein for two reasons:

[0194] 1). hFIX is a therapeutically relevant protein. Althoughtransient expression of hFIX is not clinically relevant, it would bedesirable to transiently express some other types of therapeuticproteins that do not require chronic expression.

[0195] 2) hFIX is a human protein and is thus capable of eliciting animmune response in mice.

[0196] One application of RNA injection is in the development andtesting of vaccines. An immune response to hFIX upon injection of hFIXRNA would demonstrate the proof of principle of using RNA as a vaccine.

[0197] 1. Description of Experiment and Results:

[0198] 40 micrograms of capped and polyadenylated hFIX RNA+40 microgramsof competitor RNA+800 units of RNasin were injected by tail vain into 1mouse. Result: 40 nanograms/milliliter of serum were detected by ELISAat 6 hours. This amount of hFIX is within the significant range of theELISA assay.

[0199] G. Hydrodynamic Delivery of HCV Genomic RNAs to Create an HCVMouse Model

[0200] Two groups of 6 mice were injected with:

[0201] 1) 50 micrograms of capped HCV full length genomic RNA called 90FL HCV (which also contains some uncapped RNA)+40 micrograms of cappedand polyadenylated hFIX RNA+400 units of RNasin; or

[0202] 2) a full length non-infectious HCV genomic RNA that has amutation in the replicase gene that makes it catalytically inactive(called 101 FL HCV)+40 micrograms of capped and polyadenylated hFIXRNA+400 units of RNasin.

[0203] The transcription templates for making the HCV RNAs were obtainedfrom Charles Rice and Washington University. Six hours after injectionthe mice were bled and hFIX levels are being measured to normalize forinjection efficiency. The injected HCV RNAs are expected to degraderapidly. Any RNA detected after a few days is likely to be RNA newlysynthesized during viral replication. A quantitative real time PCRmethod has been developed to measure the levels of HCV RNA in the liversof these mice. If replication of the virus occurs, then the levels ofHCV RNAs in the mice injected with 90 FL HCV will be greater than thelevels in mice injected with 101 FL HCV when measured weeks afterinjection. A histological assay is also being developed in order toassay for the synthesis of HCV proteins. Three different positiveoutcomes are possible 1) The RNA enters the liver but is not translatedand does not replicate 2) the RNA enters the liver and is translated butdoes not replicate 3) the RNA enters the liver, is translated andreplicates. All three outcomes are useful model systems. If 1, 2 or 3occurs then this system could be used to test ribozymes directed againstHCV RNAs (see experiment 9 below). If 2 or 3 occurs then, the thissystem could be used to test inhibitors of HCV translation, replicationand infection.

[0204] Injection of this RNA did not result in a viral replication cyclefor HCV. However, another group has used a similar method to initiate ahepatitis delta replication cycle. See Chang J, Sigal L J, Lerro A,Taylor J., J Virol.75(7):3469-73 (2001).

[0205] H. In vivo Cleavage of HCV RNAs by Ribozymes

[0206] DNAzymes targeting the IRES of HCV have been chemicallysynthesized. We hydrodynamically injected these ribozymes into mice andassessed their ability to decrease the levels of injected HCV RNAswithin the liver. Five nanomoles of DNAzyme targetting the IRES wascoinjected with 20 μg of an RNA comprised of the HCV IRES followed bythe firefly luciferase coding sequence followed by 30 adenosines. Thesequence of the DNAzyme was 5′-GAGGTTTAGGAGGCTAGCTACAACGATCGTGCTCA-3′(SEQ ID NO:013). Mice that received the DNAzyme in combination with thetarget RNA emitted 95% less light at 6 hours than mice that received thetarget RNA alone. Conclusion: We demonstrated that this DNAzyme caninhibit translation from the HCV IRES, presumably by cleaving the IRESRNA sequence. Synthetic ribozymes were also tested using an analogousmethodology and were found to be ineffective.

[0207] I. This experiment is to do a timecourse of luciferase expressionafter a single injection of capped and polyadenylated RNA. If thefollowing condition is met, then we can use a first order exponentialdecay fit (described by Equation 1) of the data to calculate thedegradation rate of the expressed protein. In order for this data to befit to a simple first order exponential decay, the half life of the mRNAmust be significantly less than the halflife of the protein (at least5-10 fold less). If this condition is not met, then a more complexmathematical relationship that takes into account the halflife of themRNA can be used. Another solution to this problem is to decrease thehalf life of the mRNA by making it uncapped or omiting the competitorRNA.

[0208] If we define the amount of protein at a given time (or the signalfrom the protein) as A, the amount of protein (or signal) at the firsttimepoint as Ao, the decay rate constant as k and time after the firstmeasurement as t, the equation would be of the form:

A=Ao exp ^((−kt))  (Equation 1)

[0209] 1. Description of the Experiment:

[0210] Four groups of 6 mice were injected with 20 micrograms of cappedpolyadenylated luciferase RNA+60 micrograms of uncapped competitorRNA+800 units of RNasin. At 3, 6, 9 or 24 hours, the mice were given anintraperitoneal injection of luciferin (1.5 micrograms/gram body weight)and the light emitted from the mouse was measured.

[0211] The results are provided in the table below: Hours Post LightUnits Standard Standard Error 1 3.000 530000.000 330000.000 150000.000 26.000 200000.000 88000.000 36000.000 3 9.000 110000.000 43000.00018000.000 4 24.000 1900.000 1100.000 440.000

[0212] Relative light units were plotted vs. time and the resultingcurve is fit to Equation 1. This analysis yields an apparent degradationrate consant of 0.297 hour⁻¹.

[0213] The most common method for measuring a half-life of a protein isthe following. In one approach, the protein is purified and sometimeslabeled (for example with radioactive iodine). The purified protein isinjected and at different times the animal is sampled and the amount ofprotein remaining at any given time is plotted vs. time and the curve isfit to an equation such as Equation 1. The advantage of our method isthat it does not require the in vitro synthesis or purification of theprotein.

[0214] J. We have constructed RNAs that contain regulatory regions ofthe HCV RNA controlling the translation of a protein called luciferase(referred to here as HCV luc RNA). We have also constructed DNAexpression plasmids that express similar RNAs once they enter cells(referred to here as HCV luc DNA). See FIG. 3 for diagrams of theseconstructs.

[0215] When either the HCV luc RNAs or the HCV luc DNAs are transfectedinto mice, they go to the liver and HCV luc RNAs or RNAs transcribedfrom the HCV luc DNAs are translated into luciferase protein. At varioustimes, the substrate of the luciferase protein, luciferin, is injectedinto the mice. The enzyme luciferase consumes the luciferin and makeslight in the process. The amount of light emitted from the mice isproportional to the amount of luciferase protein present at the time ofthe sampling.

[0216] We have synthesized short synthetic oligonucleotides of a typeknown as Morpholino oligos. We mixed 1 nanomol of a morpholino oligowith 10 micrograms of HCV luc RNA or 1 microgram of HCV luc DNA. Themorpholino oligo was made by Gene Tools, LLC in Corvallis, Oreg. and hasthe sequence 5′-TCTTTGAGGTTTAGGATTCGTGCTC-3′ (SEQ ID NO:14). Thismixture is then added to 1.8 milliliters of buffer and injected underhigh pressure into the tail veins of mice as described in our previousapplication. As a control, mixtures that do not contain the inhibitorare injected into other mice. In the presence of inhibitor, emittedlight is reduced by more than 90%. We conclude from this finding thattranslation of the injected RNA or translation of the RNA produced fromthe injected DNA is prevented by the inhibitor by an antisensemechanism. In the case of the injected RNA we can only follow thisinhibition for about 24 hours, because of the limited stability of theRNA in cells. In the case of the injected DNA, we can monitortranslation for about 8 days. The translational inhibition lasted forthe whole duration of the time we could measure translation in thissystem.

[0217] K.

[0218] Experiment A

[0219] control group: RNAs containing the HCV IRES and a luciferasereporter sequence are injected into mice and they glow when this RNA istranslated into luciferase protein

[0220] Test group:

[0221] Coinject inhibitor with RNA. Both go to the same cells.Inhibition is expressed as activity (glowing) compared to control group.

[0222] Experiment B

[0223] Same as experiment A except we inject a DNA that encodes thetarget RNA along with the inhibitor. The DNA goes to the nucleus of themouse hepatocytes and is transcribed to give the target RNA. This RNAgoes to the cytoplasm of the cells where it interacts with theinhibitor.

[0224] The constructs employed in these experiments are provided in FIG.4. The results of these experiments with antisense and DNAzymeinhibitors are provided in FIGS. 5A to 5F.

[0225] The above screening protocol in which the inhibitor and RNA/DNAare coadministered offers important advantages in terms of allowing oneto separate issues of drug delivery from issues of drug efficacy.

[0226] It is evident from the above results and discussion that thesubject invention provides a viable way of using RNAi agents innon-embryonic mammalian organisms, where the subject methods andcompositions can be employed for a variety of different academic andtherapeutic applications. In addition, the subject invention provides animproved method of transferring a nucleic acid into a target cell isprovided by the subject invention. Specifically, the subject inventionprovides for a highly efficient in vivo method for naked nucleic acidtransfer which does not employ viral vectors and therefore provides manyadvantages over prior art methods of nucleic acid transfer. As such, thesubject invention represents a significant contribution to the art.

[0227] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0228] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 14 1 21 RNA Artificial Sequence oligonucleotide 1 ucgaaguacucagcguaagu u 21 2 21 RNA Artificial Sequence oligonucleotide 2cuuacgcuga guacuucgau u 21 3 21 RNA Artificial Sequence oligonucleotide3 cuuacgcuga guacuucgau u 21 4 21 RNA Artificial Sequenceoligonucleotide 4 uugaaugcga cucaugaagc u 21 5 21 RNA ArtificialSequence oligonucleotide 5 agcuucauaa ggcgcaugcu u 21 6 21 RNAArtificial Sequence oligonucleotide 6 uuucgaagua uuccgcguac g 21 7 23RNA Artificial Sequence oligonucleotide 7 cugugagauc uacggagccu guu 23 823 RNA Artificial Sequence oligonucleotide 8 uugacacucu agaugccucg gac23 9 33 RNA Artificial Sequence oligonucleotide 9 ggauuccaau ucagcgggagccaccugaug aag 33 10 36 RNA Artificial Sequence oligonucleotide 10uaaccuaagg uugagucgcu cucggugggc uaguuc 36 11 66 RNA Artificial Sequenceoligonucleotide 11 ggucgaagua cucagcguaa gugaugucca cuuaaguggguguuguuugu guuggguguu 60 uugguu 66 12 78 RNA Artificial Sequenceoligonucleotide 12 gggauggacg auggccuuga ucuuguuuac cgucacacccaccacuggga gauacaagau 60 caaggccauc gucuuccu 78 13 35 DNA ArtificialSequence oligonucleotide 13 gaggtttagg aggctagcta caacgatcgt gctca 35 1425 DNA Artificial Sequence oligonucleotide 14 tctttgaggt ttaggattcgtgctc 25

What is claimed is:
 1. A method of reducing expression of a codingsequence in a target cell of a non-embryonic mammal, said methodcomprising: administering to said mammal an effective amount of an RNAiagent specific for said coding sequence to reduce expression of saidcoding sequence.
 2. The method according to claim 1, wherein said RNAiagent is an interfering ribonucleic acid.
 3. The method according toclaim 2, wherein said interfering ribonucleic acid is a siRNA.
 4. Themethod according to claim 2, wherein said interfering ribonucleic acidis a shRNA.
 5. The method according to claim 1, wherein said RNAi agentis a transcription template of an interfering ribonucleic acid.
 6. Themethod according to claim 5, wherein said transcription template is adeoxyribonucleic acid.
 7. The method according to claim 6, wherein saiddeoxyribonucleic acid encodes a shRNA.
 8. The method according to claim1, wherein said non-embryonic mammal is an adult.
 9. The methodaccording to claim 8, wherein said non-embryonic mammal is a juvenile.10. The method according to claim 1, wherein said RNAi agent ishydrodynamically administered to said non-embryonic mammal.
 11. Themethod according to claim 10, wherein said RNAi agent ishydrodynamically administered to said non-embryonic mammal inconjunction with an RNAse inhibitor.
 12. A pharmaceutical preparationcomprising an RNAi agent in a pharmaceutically acceptable deliveryvehicle.
 13. The pharmaceutical preparation according to claim 12,wherein said preparation further comprises an RNAse inhibitor.
 14. A kitfor use in practicing the method of claim 1, said kit comprising: (a) apharmaceutical preparation comprising an RNAi agent in apharmaceutically acceptable delivery vehicle; and (b) instructions forpracticing the method of claim
 1. 15. The kit according to claim 14,wherein said kit further comprises an RNAse inhibitor.
 16. Anon-embryonic non-human animal comprising an RNAi agent.
 17. Anon-embryonic non-human animal produced according to the method ofclaim
 1. 18. A method for introducing a ribonucleic acid into a targetcell of a vascularized multi-cellular organism, said method comprising:administering said ribonucleic acid as a naked ribonucleic acid into thevascular system of said organism to introduce said ribonucleic acid intosaid target cell of said vascularized multi-cellular organism.
 19. Themethod according to claim 18, wherein said administering is intravenous.20. The method according to claim 18, wherein said vascularizedmulti-cellular organism is a mammal.
 21. The method according to claim18, wherein said target cell is a hepatic cell.
 22. The method accordingto claim 18, wherein said method further comprises administering anRNAse inhibitor.
 23. The method according to claim 18, wherein saidmethod further comprises administering a competitor ribonucleic acid tosaid organism.
 24. A method for introducing a nucleic acid into a targetcell of a vascularized multi-cellular organism, said method comprising:hydrodynamically administering to said vascularized multi-cellularorganism an aqueous formulation comprising said nucleic acid as a nakednucleic acid and an RNase inhibitor to introduce said nucleic acid intosaid target cell.
 25. The method according to claim 24, wherein saidnucleic acid is a deoxyribonucleic acid.
 26. The method according toclaim 24, wherein said nucleic acid is ribonucleic acid.
 27. The methodaccording to claim 24, wherein said aqueous formulation furthercomprises competitor ribonucleic acid.
 28. A kit for use in delivering anucleic acid to a target cell of a vascularized multi-cellular organism,said kit comprising: said nucleic acid present as a naked nucleic acid;and an RNase inhibitor;
 29. The kit according to claim 28, wherein saidnucleic acid is a deoxyribonucleic acid.
 30. The kit according to claim28, wherein said nucleic acid is a ribonucleic acid.
 31. The kitaccording to claim 28, wherein said kit further comprises instructionsfor introducing said formulation into the vascular system amulti-cellular organism.
 32. The kit according to claim 28, wherein saidkit further comprises a competitor ribonucleic acid.
 33. The kitaccording to claim 28, wherein said kit further comprises a means forvascularly administrating said aqueous formulation.
 34. A method ofdetermining the activity of an RNA virus candidate modulatory agent,said method comprising: (1) hydrodynamically administering to avascularized multicellular animal: (a) a nucleic acid construct thatincludes either: (i) a RNA molecule that includes at least oneregulatory element of said RNA virus operably linked to a reporterelement; or (ii) a DNA molecule capable of being transcribed into saidRNA molecule; and (b) said candidate modulatory agent; and (2) observingthe effect of said modulatory agent on the activity of said construct isto determine the activity of said candidate modulatory agent.
 35. Themethod according to claim 34, wherein said nucleic acid method furthercomprises hydrodynamically administering an RNAse inhibitor.
 36. Themethod according to claim 34, wherein said method further comprisinghydrodynamically administering a competitor RNA.
 37. The methodaccording to claim 34, wherein said RNA virus is HCV.